The response of chick embryo hearts to muscarinic agonists prior to ingrowth of the vagus nerve is markedly decreased. Studies in our laboratory of the binding of the potent muscarinic agonist (3H)-quinuclidinylbenzilate did not reveal significant differences in the number of receptors or in binding properties of muscarinic ligands to whole heart homogenates studied prior to and after innervation. The hypothesis is presented here that the absence or block of factors that mediate the muscarinic response subsequent to agonist binding is responsible for the decreased physiologic response to muscarinic agonists prior to innervation. The test this hypothesis, studies of the response of cyclic-GMP and K ion permeability to muscarinic agonist binding in non-innervated hearts are proposed. Preliminary experiments have demonstrated the presence of muscarinic receptors in heart cell cultures. Studies are proposed to test the hypothesis that the muscarinic response properties of these cells represent those of the hearts from which cultures were derived and that these properties are stable in culture. It is further hypothesized that neuronal and neurohumoral stimuli are capable of facilitating the development of a "mature" muscarinic response in heart cell cultures. In this application, studies are proposed to determine the effects of growth of cells or aggregates in the presence of either carbamylcholine, medium conditioned by growth of chick sympathetic ganglion cells or co-culture with sympathetic ganglion cells on the physiology and biochemistry of the muscarinic response in culture.